Enteropathogenic Escherichia coli (EPEC) is a significant cause of diarrhea world-wide, with disease occuring most frequently in developing countries [1-3]. In these countries, disease occurs regularly in hospitals and clinics, as well as in the general community. EPEC outbreaks in developed countries, on the other hand, usually consist of sporadic, isolated incidents which are localized to neonatal nurseries of hospitals or day-care centers. Infants less than 6 months of age are most often affected, although EPEC is also capable of causing disease in children and adults. The transmission of EPEC infections is thought to occur primarily by the fecal-oral route as a result of contact with infected individuals or with contaminated surfaces or food. The isolation of EPEC from asymptomatic individuals has led to speculation that some individuals may be carriers who can also spread infection.
Clinical symptoms of EPEC infection in children consist of diarrhea which varies in duration (days to months) and severity [3,4]. In addition to profuse watery stool, symptoms include dehydration, fever, vomiting and weight loss. In protracted or severe cases, disease is often associated with the delayed growth of children, metabolic acidosis (decrease in blood pH resulting from a loss of bicarbonate [5,6]) and, in extreme cases, death. Adults participating in volunteer studies of EPEC infection displayed symptoms similar to those observed in children, but of shorter duration.
Results of volunteer studies indicate that, at least in adults, a relatively large infectious dose of organisms is required to produce symptoms which typically occur 7 to 16 h after infection [1,7]. For ethical reasons, similar information for EPEC infection in children is not available. It is speculated however, that a much lower number of organisms is required to infect children since transmission frequently occurs in hospitals or day-care facilities [1]. Treatment for EPEC infection usually consists of rehydration therapy and, if necessary, nutritional supplementation. Antibiotics are often used to treat EPEC infection though their overall effectiveness is uncertain [3].
Biopsies from children infected with EPEC reveal that the bacteria predominantly colonize the small intestine, although the large intestine can also be involved, presumably due to bacterial overgrowth [2,4,8,9]. The primary histopathological consequence of colonization is the atrophy or degeneration of microvilli at sites of bacterial attachment and the intimate association of bacteria with pedestal-like structures formed by host epithelial cells (e.g., enterocytes). This characteristic effect is referred to as an attaching and effacing (A/E) lesion [4,9-11]. Other features include the deterioration of the terminal web (apical region beneath microvilli consisting of cytoskeletal proteins which are physically associated with the central actin filaments of microvilli) of enterocytes, a reduction in mucosal thickness, and a general disordered arrangement of enterocytes. Bacteria are rarely found within intestinal epithelial cells or in the lamina propria, suggesting that EPEC are not invasive. An infiltration of inflammatory cells into the lamina propria of the intestine is also frequently observed during EPEC infection.
Clinical symptoms caused by EPEC result from bacterial attachment to the intestinal epithelium. Donnenberg and Kaper proposed a model in which EPEC attachment involves a three-step process [12]. The initial step consists of initial, non-intimate attachment of bacteria as microcolonies to epithelial cells. Next, the bacteria secrete several proteins which induce signal transduction pathways in epithelial cells. These signals initiate cytoskeletal rearrangement followed by the effacement of microvilli of host cells. In the final stage of attachment, cytoskeletal components are organized to form cup-like pedestal structures which partially surround adherent organisms. The latter steps of effacement and intimate attachment result in the characteristic A/E lesions associated with EPEC [11]. A modified version of this model has recently been suggested by Hicks, et al. In their model, three-dimensional microcolonies of EPEC are thought to develop after, not before, intimate attachment has occurred [13].
Since adherence is an important factor in EPEC pathogenesis, considerable research has been performed to identify bacterial and eukaryotic cell structures involved in attachment. So far, two bacterial structures have been relatively well characterized. The first, bundle-forming pili (BFP), are associated with the initial, non-intimate attachment of EPEC as microcolonies to discrete sites on epithelial cells, a pattern which is referred to as localized adherence (LA) [14,23,27]. Scanning electron micrographs of LA EPEC revealed that BFP are involved in mediating inter-bacterial linkages within microcolonies. Whether the BFP also function as adhesins for EPEC binding to epithelial cells remains to be resolved, however, since these structures appear to mediate bacterial binding to HEp-2 cells but not to human intestinal tissue in organ culture [13,27,47]. A second bacterial protein involved in attachment is intimin [36,48]. This protein is necessary for a later stage of EPEC attachment in that it focuses host cell cytoskeletal components beneath adherent bacteria to form A/E lesions [33].
Cravioto, et al. initially demonstrated that EPEC adhered to HEp-2 cells in greater numbers than other groups of E. coli studied, and that this adherence was not due to type I fimbriae [14]. Type I pili are structures which are expressed with similar frequency by pathogenic and non-pathogenic Escherichia coli (E. coli) strains, and whose binding is inhibited by mannose [15]. Subsequent investigations resulted in several different structures being proposed as EPEC adhesins. These structures included unidentified non-fimbrial [16] and fimbrial adhesins [17-19], fimbriae with N-terminal sequence homology to the fimbriae of uropathogenic and diffusely-adhering E. coli [20], and a 32 kDa outer membrane protein [21] (later reported to be OmpF [22]). However, the observation that EPEC grown in tissue culture medium attached to epithelial cells in a LA pattern [23,24], and that this phenotype was encoded by a large EPEC adherence factor (EAF) plasmid [25], led to the identification of a structure required for this pattern of binding.
In 1991, Giron, et al. described unqiue rope-like structures, termed BFP, [26] which appeared by scanning electron microscopy, to intercourse between bacteria to form microcolonies, and to attach the microcolonies to HEp-2 cells. Their role in attachment was supported by observations that antibodies raised against purified BFP partially inhibited EPEC attachment, and mutants lacking the EAF plasmid did not express BFP. The structural subunit of BFP is BfpA [52].
Following the effacement of microvilli, the formation of actin pedestals characteristic of A/E lesions requires the bacterial protein intimin [33,35,36]. Intimin is a 94 kDa outer membrane protein encoded by the eae gene of the locus of enterocyte effacement (LEE). The expression of this protein is necessary to focus host cytoskeletal proteins which accumulate beneath the organisms into pedestals, and for bacteria to become intimately associated (less than 10 mn separation) with this structure [11]. Based on serological and genetic techniques, EPEC intimins have been classified into three groups [35,37,38]. Despite variations in antigenicity and gene sequences, however, the proteins are believed to be functionally equivalent. The greatest diversity among these proteins occurs within the C-terminus, which is also thought to be the host cell binding domain [39].
Recently, Knutton, et al. demonstrated that intimin expression is down-regulated following the formation of A/E lesions [40]. This may indicate that, once bacteria have achieved their goal of becoming intimately associated with host cells, continued expression of this protein is no longer required in order to remain attached. Alternatively, since intimin is immunogenic [41], this may be a mechanism by which EPEC are able to evade the host immune response.
EPEC receptors on host eukaryotic cells are less well characterized than bacterial structures involved in attachment. Recently, the receptor for intimin, which was previously believed to be a host cell protein (Hp90), was also shown to be a protein secreted by EPEC [31-34]. This protein, referred to as Tir (translocated intimin receptor) (78 kDa) or E. coli secreted protein E (EspE) [25], is translocated into host cells where it becomes phosphorylated at tyrosine residues and then serves as the receptor for intimin [31,50]. Frankel, et al. reported that intimin may also bind to .beta..sub.1 integrins [49].
Investigations into the regulation of EPEC virulence factor expression were initiated by the observations of Vuopio-Varkila and Schoolnik that the LA phenotype of EPEC was promoted by growing the bacteria in a defined medium [24]. Since then, both environmental and genetic factors that regulate the expression of EPEC virulence factors have been described.
In the report of Vuopio-Varkila and Schoolnik, which described the positive effect of tissue culture growth medium on EPEC attachment, the increase in attachment correlated with higher levels of BFP expression [24]. Subsequently, several reports identified growth conditions that are optimal for the expression of EPEC virulence factors. Specific media components which were found to affect the expression of BFP or Esps include: calcium (BfpA and Esps), ammonium (BfpA), and FeNO.sub.3 (Esps) [44,45]. Results from our laboratory indicated that EPEC binding to HEp-2 cells and the expression of BfpA and intimin were also affected by carbon source [42]. Regarding general environmental conditions, the secretion of Esps was dependent on the osmolarity and pH of the tissue culture medium [43,44,75]. Growth at 37.degree. C. was also optimal for the expression of BfpA and Esps, and for the formation of A/E lesions [44-46]. Since the expression of different EPEC virulence factors is affected by the same environmental condition(s), these factors may be coordinately regulated [44].
Studies by several research groups have implicated carbohydrate structures as host cell receptors for EPEC. In studies where soluble compounds were used to inhibit attachment, N-acetyl-galactosamine [21], GM.sub.3 gangliosides [27], or fucosylated tetra- and pentasaccharides [28] and the GalNAc.beta.(1.fwdarw.4)Gal portion of asialo-GM.sub.1 and asialo-GM.sub.2 structures [2] were found to be implicated in EPEC attachment to eukaryotic cells, based on their inhibition of EPEC binding (Table 1). These structures, excluding GM.sub.3 gangliosides, presumably inhibited initial attachment since inhibition was measured as a decrease in the numbers of LA EPEC bound to epithelial cells. Alternatively, when glycolipids were layered onto thin layer chromatography plates, EPEC preferentially recognized the GalNAc.beta.(1.fwdarw.4)Gal portion of asialo-GM.sub.1 and asialo-GM.sub.2 sequences [29]. These studies also suggested that the sequences were involved in initial attachment, since LA-negative mutants did not bind to these sequences. In addition to these results, work performed in our laboratory suggested that EPEC recognize lactosamine sequences on eukaryotic cells. We examined EPEC LA to Chinese hamster ovary cells or mutants of these cells which express altered oligosaccharide structures on their surface. Results of these studies suggested that asialo-lactosamine sequences on N-linked glycoproteins were sufficient for EPEC binding. Our results also supported a role for O-linked glycoproteins or glycolipids in attachment [30].
TABLE 1 Oligosaccharide Sequences Proposed to be Involved in EPEC Binding Oligosaccharide .sup.a Inhibitory Sequence N-acetylgalactosamine GalNAc [21] difucosyllactose Fuc.alpha.(1.fwdarw.2)Gal.beta.(1.fwdarw.4)[Fuc.alpha.(1.fwdarw.3)]Glc [28] lacto-N-fucopentaose Fuc.alpha.(1.fwdarw.2)Gal.beta.(1.fwdarw.3)GlcNAc.beta.(1.fwdarw.3)Gal.bet a.(1.fwdarw.4)Glc isomers Gal.beta.(1.fwdarw.3)[Fuc.alpha.(1.fwdarw.4)]GlcNAc.beta.(1.fwdarw.3)Gal.b eta.(1.fwdarw.4)Glc; Gal.beta.(1.fwdarw.4)[Fuc.alpha.(1.fwdarw.3)]GlcNAc.beta.(1.fwdarw.3)Gal.b eta.(1.fwdarw.4)Glc [28] asialo GM.sub.1 .sup.b Gal.beta.(1.fwdarw.3)GalNAc.beta.(1.fwdarw.4)Gal.beta.(1.fwdarw.4)Glc.beta .(1.fwdarw.1)cer [29] asialo GM.sub.2 .sup.b GalNAc.beta.(1.fwdarw.4)Gal.beta.(1.fwdarw.4)Glc(1.fwdarw.1)cer [29] GM.sub.3 Sia.alpha.(2.fwdarw.3)Gal.beta.(1.fwdarw.4)Glc.beta.(1.fwdarw.1)cer [27] lactosamine of N-linked Gal.beta.(1.fwdarw.3,4)GlcNAc [30] glycoproteins O-linked glycoproteins not known [30] or glycolipids .sup.a N-acetylgalactosamine = GalNAc; fucose = Fuc; galactose = Gal; N-acetylglucosamine = GlcNac; glucose = Glc; Sia = sialic acid; ceramide = cer .sup.b Underlined portion of sequence proposed to be inhibitory
Several reports suggest that EPEC recognize lactosyl structures on epithelial cells. However, additional carbohydrate groups (i.e., sialic acid and fucose) are frequently attached to these core structures. While our previous results using CHO cell Lec mutants indicated that EPEC do not require sialic acid in order to bind, the importance of fucose in these interactions was not addressed since CHO cells do not express certain fucosylated glycans [51].
In view of the above, there is a need for a compound which would treat diarrhea and related symptoms caused by EPEC. A preferred compound would be administered noninvasively, such as orally, and would attenuate the virulence of EPEC organisms which express virulence factors such as BFP and intimin.